This invention was made with the support of the United States Government and the United States Government has certain rights in the invention.
The present invention relates to the prophylaxis and treatment of anthrax infections and, more particularly, to compounds that act as specific inhibitors of Anthrax Lethal Factor activity, methods and means for making such inhibitors and their use as pharmaceuticals.
Anthrax is a zoonotic illness recognized since antiquity. In the 1870s, Robert Koch demonstrated for the first time the bacterial origin of a specific disease, with his studies on experimental anthrax, and also discovered the spore stage that allows persistence of the organism in the environment. Shortly afterward, Bacillus anthracis was recognized as the cause of woolsorter disease (inhalational anthrax). William Greenfield""s successful immunization of livestock against anthrax soon followed in 1880, although Louis Pasteur""s 1881 trial of a heat-cured anthrax vaccine in sheep is usually remembered as the initial use of a live vaccine.
Human cases of anthrax are invariably zoonotic in origin, with no convincing data to suggest that human-to-human transmission has ever taken place. Primary disease takes one of three forms: (1) Cutaneous, the most common, results from contact with an infected animal or animal products; (2) Inhalational is much less common and a result of spore deposition in the lungs, while (3) Gastrointestinal is due to ingestion of infected meat. Most literature cites cutaneous disease as constituting the large majority (up to 95%) of cases.
Bacillus anthracis is a large, gram-positive, sporulating rod, with square or concave ends. Growing readily on sheep blood agar, B. anthracis forms rough, gray-white colonies of four to five mm, with characteristic comma-shaped or xe2x80x9ccomet-tailxe2x80x9d protrusions. Several tests are helpful in differentiating B. anthracis from other Bacillus species. Bacillus anthracis is characterized by an absence of the following: Hemolysis, motility, growth on phenylethyl alcohol blood agar, gelatin hydrolysis, and salicin fermentation. Bacillus anthracis may also be identified by the API-20E and API-50CHB systems used in conjunction with the previously mentioned biochemical tests. Definitive identification is based on immunological demonstration of the production of protein toxin components and the poly-D-glutamic acid capsule, susceptibility to a specific bacteriophage, and virulence for mice and guinea pigs. The virulence of B anthracis is dependent on two toxins, lethal toxin and edema toxin, as well as on the bacterial capsule. The importance of a toxin in pathogenesis was demonstrated in the early 1950s, when sterile plasma from anthrax-infected guinea pigs caused disease when injected into other animals (Smith, H. and J. Keppie, Nature 173:869-870 (1954)). It has since been shown that the anthrax toxins are composed of three entities, which in concert lead to some of the clinical effects of anthrax (Stanley, J. L. and H. Smith, J. Gen Microbiol 26:49-66 (1961); Beall, F. A. et al., J. Bacteriol 83:1274-1280 (1962)). The first of these, protective antigen, is an 83 kd protein so named because it is the main protective constituent of anthrax vaccines. The protective antigen binds to target cell receptors and is then proteolytically cleaved of a 20 kd fragment. A second binding domain is then exposed on the 63 kd remnant, which combines with either edema factor, an 89 kd protein, to form edema toxin, or lethal factor, a 90 kd protein, to form lethal toxin (Leppla, S. H. et al., Salisbury Med Bull Suppl., 68:41-43 (1990)). The respective toxins are then transported across the cell membrane, and the factors are released into the cytosol where they exert their effects. Edema factor, a calmodulin-dependent adenylate cyclase, acts by converting adenosine triphosphate to cyclic adenosine monophosphate. Intracellular cyclic adenosine monophosphate levels are thereby increased, leading to the edema characteristic of the disease (Leppla, S. H., Proc Natl Acad Sci USA 79:3162-3166 (1982)). The action of lethal factor, believed to be a metalloprotease, is less well understood. Lethal toxin has been demonstrated to lyse macrophages at high concentration, while inducing the release of tumor necrosis factor and interleukin 1 at lower concentrations (Hanna, P. C. et al., Proc Natl Acad Sci USA 90:10198-10201 (1993); Freidlander, A. M., J Biol Chem. 261:7123-7126 (1986)).
It has been shown that a combination of antibodies to interleukin 1 and tumor necrosis factor was protective against a lethal challenge of anthrax toxin in mice, as was the human interleukin 1 receptor antagonist (Hanna, P. C. et al., Proc Natl Acad Sci USA 90:10198-10201 (1993)). Macrophage-depleted mice were shown to resist lethal toxin challenge, but to succumb when macrophages were reconstituted. The genes for both the toxin and the capsule are carried by plasmids, designated pX01[33] and pX02, respectively (Green, B. D. et al., Bacillus anthracis Infect Immunol. 49:291-297 (1985); Uchida, I. Et al., J Gen Microbiol. 131:363-367 (1985)).
Disease occurs when spores enter the body, germinate to the bacillary form, and multiply. In cutaneous disease, spores gain entry through cuts, abrasions, or in some cases through certain species of biting flies. Germination is thought to take place in macrophages, and toxin release results in edema and tissue necrosis but little or no purulence, probably because of inhibitory effects of the toxins on leukocytes. Generally, cutaneous disease remains localized, although if untreated it may become systemic in up to 20% of cases, with dissemination via the lymphatics. In the gastrointestinal form, B. anthracis is ingested in spore-contaminated meat, and may invade anywhere in the gastrointestinal tract. Transport to mesenteric or other regional lymph nodes and replication occur, resulting in dissemination, bacteremia, and a high mortality rate. As in other forms of anthrax, involved nodes show an impressive degree of hemorrhage and necrosis.
The pathogenesis of inhalational anthrax is more fully studied and understood. Inhaled spores are ingested by pulmonary macrophages and carried to hilar and mediastinal lymph nodes, where they germinate and multiply, elaborating toxins and overwhelming the clearance ability of the regional nodes. Bacteremia occurs, and death soon follows. Penicillin remains the drug of choice for treatment of susceptible strains of anthrax, with ciprofloxacin and doxycycline employed as suitable alternatives. Some data in experimental models of infection suggest that the addition of streptomycin to penicillin may also be helpful. Penicillin resistance remains extremely rare in naturally occurring strains; however, the possibility of resistance should be suspected in a biological warfare attack. Cutaneous anthrax may be treated orally, while gastrointestinal or inhalational disease ordinarily should receive high doses of intravenous antibiotics (penicillin G, 4 million units every 4 hours; ciprofloxacin, 400 mg every 12 hours; or doxycycline hyclate, 100 mg every 12 hours). The more severe forms require intensive supportive care and have a high mortality rate despite optimal therapy. The use of anti-anthrax serum, while no longer available for human use except in the former Soviet Union, was thought to be of some use in the preantibiotic era, although no controlled studies were performed.
Although anthrax vaccination dates to the early studies of Greenfield and Pasteur, the xe2x80x9cmodernxe2x80x9d era of anthrax vaccine development began with a toxin-producing, unencapsulated (attenuated) strain in the 1930s. Administered to livestock as a single dose with a yearly booster, the vaccine was highly immunogenic and well tolerated in most species, although somewhat virulent in goats and llamas. This preparation is essentially the same as that administered to livestock around the world today. The first human vaccine was developed in the 1940s from nonencapsulated strains. This live spore vaccine, similar to Sterne""s product, is administered by scarification with a yearly booster. Studies show a reduced risk of 5- to 15-fold in occupationally exposed workers (Shlyakhov, E. N and E. Rubenstein, Vaccine 12:727-730 (1994)).
British and U.S. vaccines were developed in the 1950s and early 1960s, with the U.S. product an aluminum hydroxide-adsorbed, cell-free culture filtrate of an unencapsulated strain (V770-NP1-R), and the British vaccine an alum-precipitated, cell-free filtrate of a Sterne strain culture. The U.S. vaccine has been shown to induce high levels of antibody only to protective antigen, while the British vaccine induces lower levels of antibody to protective antigen but measurable antibodies against lethal factor and edema factor (Turnbull, P. C. B. et al., Infect Immunol. 52:356-363 (1986); Turnbull, P. C. B. et al., Med Microbiol Immunol. 177:293-303 (1988)). Neither vaccine has been examined in a human clinical efficacy trial. A high number of the recipients of the vaccine have reported some type of reaction to vaccination. The preponderance of these events were minor. Manufacturer labeling for the current Michigan Department of Public Health anthrax vaccine adsorbed (AVA) product cites a 30% rate of mild local reactions and a 4% rate of moderate local reactions with a second dose. The current complex dosing schedule for the AVA vaccine consists of 0.5 mL administered subcutaneously at 0, 2, and 4 weeks, and 6, 12, and 18 months, followed by yearly boosters. Animal studies examining the efficacy of available anthrax vaccines against aerosolized exposure have been performed. While some guinea pig studies question vaccine efficacy, primate studies have support its role. In recent work, rhesus monkeys immunized with 2 doses of the AVA vaccine were challenged with lethal doses of aerosolized B anthracis spores. All monkeys in the control group died 3 to 5 days after exposure, while the vaccinated monkeys were protected up to 2 years after immunization (Ivins, B. E. et al., Salisbury Med Bull Suppl. 87:125-126 (1996)). Another trial used the AVA vaccine in a 2-dose series with a slightly different dosing interval, and again found it to be protective in all rhesus monkeys exposed to lethal aerosol challenge (Pitt, M. L. M. et al., Salisbury Med Bull Suppl. 87:130 (1996)) Thus, available evidence suggests that two doses of the current AVA vaccine should be efficacious against an aerosol exposure to anthrax spores. In addition, a highly purified, minimally reactogenic, recombinant protective antigen vaccine has been investigated, using aluminum as well as other adjuvants. Other approaches include cloning the protective antigen gene into a variety of bacteria and Viruses, and the development of mutant, avirulent strains of B anthracis. One significant limitation on the use of vaccines is that existing vaccines provide no protection against a number of strains of B. anthracis. 
Recent incidents, such as the suspected use of biological and chemical weapons during the Persian Gulf War, underscore the threat of biological warfare either on the battlefield or by terrorists. Anthrax has been the focus of much attention as a potential biological warfare agent for at least six decades, and modeling studies have shown the potential for use in an offensive capacity. Dispersal experiments with the simulant Bacillus globigii in the New York subway system in the 1960s suggested that release of a similar amount of B anthracis during rush hour would result in 10,000 deaths. On a larger scale, the World Health Organization estimated that 50 kg of B anthracis released upwind of a population center of 500,000 would result in up to 95,000 fatalities, with an additional 125,000 persons incapacitated (Huxsoll, D. L. et al., JAMA 262:677-679 (1989)). Both on the battlefield and in a terrorist strike, B. anthracis has the attribute of being potentially undetectable until large numbers of seriously ill individuals present with characteristic signs and symptoms of inhalational anthrax. Given these findings, efforts to prevent the disease or to ameliorate or treat its effects are of obvious importance. The U.S. military""s current M17 and M40 gas masks provide excellent protection against the 1 to 5 xcexcm particulates needed for a successful aerosol attack. Assuming a correct fit, these masks would be highly effective if in use at the time of exposure. Some protection might also be afforded by various forms of shelter. The preexposure use of the current AVA anthrax vaccine, which is approved by the U.S. Food and Drug Administration, appears to be an important adjunct. Results of primate studies also support the concept of postexposure antibiotic prophylaxis. One study showed that 7 of 10 monkeys given penicillin, 8 of 9 given ciprofloxacin, 9 of 10 treated with doxycycline, and all 9 receiving doxycycline plus postexposure vaccination survived a lethal aerosol challenge, with all animals receiving antibiotics for 30 days following exposure (Friedlander, A. M. et al., J Infect Dis. 167:1239-1242 (1993). Earlier research suggested that short courses of prophylactic antibiotics delayed but did not prevent clinical disease (Henderson, D. W. et al., J Hyg. 54:28-36 (1956). Accordingly, in the event of documented exposure, prolonged prophylactic antibiotic use, as well as vaccination, would be mandatory. In the biological warfare setting, the differential diagnosis of inhalational anthrax would include plague and tularemia. Fluoroquinolones also have activity against these diseases, supporting the use of ciprofloxacin and perhaps other drugs of this class as either a preexposure or postexposure measure.
It is therefore apparent that while certain prophylactic and treatment schemes may prove useful in preventing or ameliorating anthrax infections, there remains a compelling need to improve the arsenal of techniques and agents available for this purpose.
Accordingly, it is an object of the invention described herein to provide compositions that are capable of precisely targeting acute responses to Anthrax Lethal Factor without producing significant undesirable side effects.
This and other objects will be apparent from consideration of the specification as a whole.
The present invention provides methods, compounds and compositions for treating anthrax infections by inhibiting anthrax lethal factor activity. In one aspect, the invention provides a compound in accordance with the formula: 
Wherein
R1, R2, R3 and R4 are each independently selected from
H, hydroxyl, alkoxy, alkylthio,
small alkyl (C1-C10) (optionally C1-C6) (optionally substituted with alkyl, cycloalkyl fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR6, OCxe2x95x90OR6, Cxe2x95x90Oxe2x80x94OR7, or Cxe2x95x90Oxe2x80x94NR8R9),
phenyl and mono and disubstituted (at positions 3 and 4) phenyl (wherein the phenyl ring is independently substituted with alkyl, trifluoromethyl, mono and di halogen atoms, alkylthio, alkoxy, nitro, cyano, morphilino, cyclohexyl, phenyl, phenolic, dioxymethylene, nitro, acetylamino);
heteroaryl (optionally substituted with alkyl, halogen, alkylthio, alkoxy, or nitro),
cycloalkyl (C3-C10) (optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, OCxe2x95x90OR5, Cxe2x95x90Oxe2x80x94OR6, or Cxe2x95x90Oxe2x80x94NR7R8)
alkenyl (C1-C10) (optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, OCxe2x95x90OR5, Cxe2x95x90Oxe2x80x94OR6, or Cxe2x95x90Oxe2x80x94NR7R8)
alkadienyl (C1-C10) (optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, xe2x80x94OCxe2x95x90OR5, xe2x80x94Cxe2x95x90Oxe2x80x94OR6, Cxe2x95x90Oxe2x80x94NR7R8)
cycloalkenyl (C4-C10), optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, xe2x80x94OCxe2x95x90OR5, xe2x80x94Cxe2x95x90Oxe2x80x94OR6, Cxe2x95x90Oxe2x80x94NR7R8
bicycloalkyl (C5-C12), optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, xe2x80x94OCxe2x95x90OR5, xe2x80x94Cxe2x95x90Oxe2x80x94OR6, Cxe2x95x90Oxe2x80x94NR7R8
tricycloalkyl (C8-C14), optionally substituted with alkyl, fluoro, aryl, heteroaryl, alkylthio, arylthio, cyano, OR5, xe2x80x94OCxe2x95x90OR5, xe2x80x94Cxe2x95x90Oxe2x80x94OR6, Cxe2x95x90Oxe2x80x94NR7R8
where
R5 is
alkyl (C1-C10), optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl, cyano, aryloxy, cycloalkyl;
aryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, cyano, aryloxy;
heteroaryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, cyano, aryloxy;
cycloalkyl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl, cyano:
R6 is
alkyl (C1-C10), optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl;
aryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
heteroaryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
cycloalkyl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl;
R7 is
alkyl (C1-C10), optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl;
aryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
heteroaryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
cycloalkyl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl; and
R8 is
alkyl (C1-C10), optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl;
aryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
heteroaryl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio;
cycloalkyl, optionally substituted with alkyl, keto, fluoro, alkoxy, alkylthio, aryl, heteroaryl.
Such compounds and compositions will be found suitable for use as specific inhibitors of anthrax lethal factor activity for the prophylaxis and treatment of anthrax infections.